Osteoporosis is a disease of progressive bone loss, leading to weak and fracture prone bones. This disease affects ~12 million Americans and is a major medical and economic burden on society. Osteoporosis is primarily a genetic disorder with fracture-related traits, such as bone mineral density (BMD), being among the most highly heritable disease associated phenotypes. In humans and mice, genetic studies to date have focused almost exclusively on the analysis of BMD. However, BMD is a complex organismal-level trait that is influenced by a complicated milieu of genetic and environmental factors. This has hampered our ability to precisely identify the causal genes that underlie genetic associations. As an alternative, we propose to focus exclusively on the genetics of a more 'simple' cell-level process, osteoblast-mediated bone formation. The objective of this proposal is to identify genes affecting osteoblast function. This will be accomplished using a novel and innovative mouse genetic reference population termed the Collaborative Cross (CC). In a pilot study, we identified two genome-wide associations in the mouse for osteoblast activity and using bone expression and human GWAS we have identified three candidate genes potentially underlying these associations. In Aim 1, we will evaluate the effect of modulating expression levels of these candidates on osteoblast function in vitro. In Aim 2, we will map additional high-resolution quantitative trait loci (QTL) for mineralized nodule formation, a physiologically relevant measure of osteoblast-mediated bone formation, in the CC. In Aim 3, we will move from QTL to the identification and validation of candidate genes for mineralized nodule formation QTL. This will be accomplished by exploiting the unique genetic aspects of the CC to bioinformatically narrow these loci, followed by RNA-seq/expression QTL studies to further pinpoint causative genes. Candidate genes for mineralized nodule formation QTL will be tested by gene overexpression and knockdown and mutant mouse studies. We expect that the study of a cell-level process will provide the means to more efficiently go from locus to gene to mechanism. Genes that we identify will serve as potential therapeutic targets capable of increasing bone formation in the setting of osteoporosis.